Genomic DNA provides the template for the information that allows the generation of proteins which are expressed and made by an organism. These proteins are generally essential for the survival of any specific cell in an organism. Therefore, the organism requires the template to be correct and free of mistakes in order to generate a protein that is functional in a cell. If a single nucleotide of this DNA sequence is mutated (a "point mutation"), the protein may be nonfunctional. Point mutations which elicit disease states are known for many proteins. Examples include sickle cell anemia hypoxanthine phosphotransferase, and p53, a tumor suppressor gene, and several oncogenes and cancer genes.
A review by Cotton, Biochem. J. 263: 1 (1989), compared several methodologies for detection of point mutations with respect to the DNA type used, the DNA stage achieved, whether the mutation position was detected, the percentage of mutations detected, the time and cost requirements, and toxicity problems. Each of the methodologies examined by Cotton presents drawbacks. DNA sequencing, for example, is time consuming and expensive. Restriction enzymes do not define the mutation position and detect less than 50% of mutations. Denaturing gradient gels and SSCP, see Murakami et al., Cancer Res. 51:3356 (1981), do not define the mutation position and are not efficient at detecting mutations. S1 nuclease and RNAse are not efficient at detecting mutations. Finally, Carbodi-imide/ABC nuclease and carbodi-imide are efficient but generate false positives and are toxic.
Recently, point mutations have been detected with the E. coli repair enzyme mutY. See Hsu et ai., Carcinogenesis 15(8): 1657 (1994). In this method a wild type labeled probe is generated using the polymerase chain reaction (PCR) described, for example, by Saiki et al., Nature 324: 163 (1986). The probe then is hybridized to the unknown sample DNA wherein mutY then cleaves mismatches when an adenosine which does not form watson crick base pairing with a guanine nucleotide. The position of mutY cleavage at A/G sites can then be determined by gel electrophoresis. This methodology is limited by the use of PCR, which itself generates mutations in the amplified DNA. See Loeb et al., Nucleic Acids & Molec. Biol. 1: 157 (1987); Tindal et al., Biochemistry 27:6008 (1988); Kunkel, loc. cit. 29:8004 (1990) .
Accordingly, there is a need for an accurate and efficient method of detecting point mutations using unamplified DNA source molecules. In addition, such a method would save time, require minimal equipment and is less expensive, as well decreasing the hazard of toxic chemicals. Also, methods of amplifying limiting amounts of the mutated sequences would have advantages.
For the same reasons, there is a need for accurate and inexpensive methods to detect non-mutated target polynucleotides from unamplified DNA source molecules.
Currently there are several amplification methodologies, well known to those skilled in the art, for the detection of non-mutated DNA. Among these techniques are the polymerase chain reaction (PCR), the ligase chain reaction (LCR), nucleic acid system-based amplification (NASBA), and cycling probe technology (CPT). Other amplification methods are well known to those skilled in the art.
The polymerase chain reaction described, for example, in U.S. Pat. No. 4,362,195, is the best known amplification system, but it is limited by the level of amplification (.about.2.2.times.10.sup.5), is prone to the generation of mutations, and can generate false positives by the generation of amplified molecules contaminating the environment. Despite these limitations, PCR is widely used in the research community. It still is not approved by governmental regulators for clinical and diagnostic applications, however.
CPT technology was developed, in part to overcome the limitations of PCR. See, for example, U.S. Pat. Nos. 4,876,187 and No. 5,011,769. The CPT technology entails the use of a synthetic molecule with two non-complementary nucleic acid sequences joined by a scissile linkage. CPT technology works by observing a hybridization event with a sample nucleic acid by a single cleavage event. This technology utilizes both the enzymatic features of RNAse H and a synthetic DNA-RNA-DNA oligonucleotide. RNAse H specifically cleaves the RNA moiety of the DNA-RNA-DNA oligonucleotide only when it is perfectly hybridized to a complementary DNA target molecule. A high concentration of the DNA-RNA-DNA molecule is converted to cleaved fragments, which are assayed by gel electrophoresis. The level of cleavage indicates the amount of target molecules present in the sample.
The CPT system does not amplify the target, alleviating the accumulation of molecules that in turn become amplifiable and generate false-positives, as occurs in PCR. The CPT technology is linear, in that increasing amounts of target DNA generate linearly more cleaved DNA-RNA-DNA oligonucleotide. (PCR generates exponentially more signal in response to the presence of more target DNA, making quantitation more problematic). Additionally, CPT can amplify up to 10.sup.6 cleaved DNA-RNA-DNA probe molecules in about 30 minutes. CPT does not generate more of the target molecule. Therefore, it does not jeopardize the laboratory environment by the possible accumulation of synthesized target DNA molecules, which in turn generate false positive results. It also is isothermal, i.e., it does not require the use of expensive automated thermocycling equipment. Further, it has been shown to detect a single molecule. The CPT technology is limited, however, because the cleavable portion of the molecule is an RNA moiety.